US8546337B2 - Self-assembling peptide nanoparticles useful as vaccines - Google Patents

Self-assembling peptide nanoparticles useful as vaccines Download PDF

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US8546337B2
US8546337B2 US12/864,576 US86457609A US8546337B2 US 8546337 B2 US8546337 B2 US 8546337B2 US 86457609 A US86457609 A US 86457609A US 8546337 B2 US8546337 B2 US 8546337B2
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Peter Burkhard
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Definitions

  • the present invention relates to self-assembling peptide nanoparticles incorporating B-cell epitopes and/or T-cell epitopes. Furthermore, the invention relates to the use of such nanoparticles for vaccination.
  • the adaptive immune system has two different responses, the humoral immune response and the cellular immune response.
  • the first is characterized by an antibody response in which these antibodies bind to surface epitopes of pathogens while the latter is characterized by cytotoxic T-lymphozytes (CTLs) that kill already infected cells.
  • CTLs cytotoxic T-lymphozytes
  • Both immune responses are further stimulated by T-helper cells that activate either the B-cells that are producing specific pathogen binding antibodies or T-cells that are directed against infected cells.
  • the specificity of the interaction between the antibodies produced by B-cells and the pathogen is determined by surface structures of the pathogen, so called B-cell epitopes, while the specificity of the interaction of CTLs with the infected target cell is by means of T-cell epitopes presented on surface molecules of the target cell, the so-called major histocompatibility complex class I molecules (MHC I).
  • MHC I major histocompatibility complex class I molecules
  • CTL-epitopes are fragments of the proteins from the pathogen that are produced by the infected cell.
  • the specificity of the interaction of the T-helper cells with the respective B-cell or CTL is determined by binding of receptor molecules of the T-helper cells to the other type of T-cell epitopes (HTL-epitopes) presented by the MHC class II molecules (MHC II) on the B-cells or CTL-cells.
  • HTL-epitopes T-cell epitopes
  • MHC II MHC class II molecules
  • Binding of the antibodies to the B-cell epitopes requires the B-cell epitope to assume a particular three-dimensional structure, the same structure that this B-cell epitope has in its native environment, i.e. when it is on the surface of the pathogen.
  • the B-cell epitope may be composed of more than one peptide chain and is organized in a three dimensional structure by the scaffold of the protein.
  • T-cell epitopes do not require a particular three-dimensional structure, rather they are bound by the respective MHC I or MHC II molecule in a very specific manner.
  • CTL epitopes are trimmed to a size of 9 amino acids in length for optimal presentation by the MHC I molecules, while HTL epitopes make a similar interaction with the MHC II molecules but may be longer than just 9 amino acids.
  • the binding of the epitopes to the MHC molecules follows very particular rules, i.e. only peptides with specific features will be able to bind to the respective MHC molecule and hence be useful as epitopes.
  • VLPs Virus-like particles
  • SAPN self-assembling peptide nanoparticles
  • the B-cell epitopes were attached at the end of the oligomerization domains in order to guarantee that the B-cell epitopes are presented at the surface of the nanoparticles in multiple copies.
  • One of the most frequently encountered protein oligomerization motif is the coiled-coil structural motif and this motif can efficiently be used in the design of these SAPN.
  • the invention relates to self-assembling peptide nanoparticles (SAPN) incorporating T-cell epitopes and/or B-cell epitopes.
  • SAPN self-assembling peptide nanoparticles
  • nanoparticles of the invention consist of aggregates of a continuous peptidic chain comprising two oligomerization domains connected by a linker segment wherein one or both oligomerization domains is a coiled-coil that incorporates T-cell epitopes and/or B-cell epitopes within its peptide sequence.
  • the invention further relates to a method of vaccinating humans or non-human animals using such self-assembling peptide nanoparticles incorporating T-cell epitopes and/or B-cell epitopes.
  • FIG. 1 The structure of mouse MHC II molecule I-Ad covalently linked to an ovalbumin peptide (OVA323-339), which is a HTL epitope for I-Ad.
  • OVA323-339 ovalbumin peptide
  • the MHC II protein is shown from the top in a C-alpha trace in gray.
  • the two helices forming the walls of the epitope binding sites are flanking the bound peptide.
  • the peptide is shown in an all-atom ball-and-stick model in black.
  • the peptide HTL epitope in its bound form is in extended conformation as can be seen more clearly by the structure of the peptide alone at the bottom of the figure.
  • FIG. 2 Schematic drawing of “even units” for trimeric and pentameric oligomerization domains [left side, A)] and trimeric and tetrameric oligomerization domains [right side, B)], respectively.
  • the number of monomers (building blocks) is defined by the least common multiple (LCM) of the oligomerization states of the two oligomerization domains D1 and D2 of the building blocks.
  • LCM least common multiple
  • the linker segments of all building blocks will be arranged as closely to each other as possible, i.e. as close to the center of the peptidic nanoparticle as possible and hence the even units will self-assemble to a spherical nanoparticle.
  • FIG. 3 Internal symmetry elements of the dodecahedron/icosahedron.
  • the rotational symmetry axes (2-fold, 3-fold and 5-fold) are displayed as lines marked 2, 3 and 5.
  • A) a monomeric building block composed of oligomerization domain D1 (left, coiled-coil domain with three-fold symmetry), a linker segment L (bottom), and oligomerization domain D2 (right; coiled-coil domain with five-fold symmetry) is displayed such that the internal symmetry elements of the oligomerization domains D1 and D2 are superimposed onto the symmetry elements of the polyhedron.
  • the complete coiled-coil domains D1 and D2 are displayed.
  • the additional symmetry objects generated by the 3-fold and the 5-fold rotational symmetry elements of the polyhedron are displayed as cylinders while the original molecule is displayed as a helix as in A).
  • FIG. 4 Dynamic lights scattering (DLS, A) and transmission electron microscopy (TEM, B) of the self-assembled peptide nanoparticles formed from peptides with the sequence SEQ ID NO:8, Example 1.
  • the DLS analysis shows size distribution with an average particle diameter of 32.01 nm and polydispersity index of 12.9% (A).
  • the TEM pictures (B) show nanoparticles of the same size as determined by DLS.
  • FIG. 5 Transmission electron microscopy (TEM) of the self-assembled peptide nanoparticles formed from peptides with the sequence SEQ ID NO:10, Example 2.
  • the TEM picture shows nanoparticles of the same size of 25 nm.
  • FIG. 6 Transmission electron microscopy (TEM) of the self-assembled peptide nanoparticles formed from peptides with the sequence SEQ ID NO:12, Example 3.
  • the TEM picture shows nanoparticles of the size of about 20 to 30 nm.
  • FIG. 7 Transmission electron microscopy (TEM) of the self-assembled peptide nanoparticles formed from peptides with the sequences SEQ ID NO:37 (panel A) and SEQ ID NO:38 (panel B), for a human and a chicken influenza vaccine, respectively (Example 9).
  • the TEM pictures show nanoparticles of the size of about 25 nm.
  • FIG. 8 Transmission electron microscopy (TEM) of the self-assembled peptide nanoparticles formed from peptides with the sequence SEQ ID NO:41, Example 11.
  • the TEM picture shows nanoparticles of the size of about 25 nm.
  • SAPN Self-assembling peptide nanoparticles
  • D1 is a synthetic or natural peptide having a tendency to form oligomers (D1) m of m subunits D1
  • D2 is a synthetic or natural peptide having a tendency to form oligomers (D2) n of n subunits D2
  • m and n each is a figure between 2 and 10, with the proviso that m is not equal n and not a multiple of n, and n is not a multiple of m
  • L is a bond or a short linker chain selected from optionally substituted carbon atoms, optionally substituted nitrogen atoms, oxygen atoms, sulfur atoms, and combinations thereof; either D1 or D2 or both D
  • a peptide is a chain or sequence of amino acids covalently linked by amide bonds.
  • the peptide may be natural, modified natural, partially synthetic or fully synthetic. Modified natural, partially synthetic or fully synthetic is understood as meaning not occurring in nature.
  • the term amino acid embraces both naturally occurring amino acids selected from the 20 essential natural ⁇ -L-amino acids, synthetic amino acids, such as ⁇ -D-amino acids, 6-aminohexanoic acid, norleucine, homocysteine, or the like, as well as naturally occurring amino acids which have been modified in some way to alter certain properties such as charge, such as phoshoserine or phosphotyrosine, or the like.
  • the amino group forming the amide bond is alkylated, or a side chain amino, hydroxy or thio functions is alkylated or acylated, or a side chain carboxy function is amidated or esterified.
  • a short linker chain L is selected from optionally substituted carbon atoms, optionally substituted nitrogen atoms, oxygen atoms, sulfur atoms, and combinations thereof, with preferably 1 to 60 atoms, in particular 1 to 20 atoms in the chain.
  • Such a short linker chain is, e.g. a polyethylenoxy chain, a sugar chain or, preferably, a peptide chain, e.g. a peptide chain consisting of 1 to 20 amino acids, in particular 1 to 6 amino acids.
  • n and n each is a figure between 2 and 10, with the proviso that m is not equal n and not a multiple of n, and n is not a multiple of m.
  • Preferred combinations of n and m are combinations wherein m is 2 and n is 5, or m is 3 and n is 4 or 5, or m is 4 and n is 5.
  • preferred combinations of n and m are combinations wherein m is 5 and n is 2, or m is 4 or 5 and n is 3, or m is 5 and n is 4. Most preferred are combinations wherein m or n is 5.
  • a coiled-coil is a peptide sequence with a contiguous pattern of mainly hydrophobic residues spaced 3 and 4 residues apart, which assembles to form a multimeric bundle of helices, as will explained in more detail hereinbelow.
  • a coiled-coil that incorporates T-cell and/or B-cell epitopes means that the corresponding epitope is comprised within an oligomerization domain such that the amino acid sequences at the N-terminal and the C-terminal ends of the epitope force the epitope to adapt a conformation which is still a coiled-coil in line with the oligomerization properties of the oligomerization domain comprising the epitope.
  • “incorporated” excludes a case wherein the epitope is attached at either end of the coiled-coil oligomerization domain.
  • T-cell epitopes shall be used to refer to both CTL and HTL epitopes.
  • T-cell epitopes bind to the MHC molecules in extended conformation ( FIG. 1 ). Therefore, incorporating T-cell epitopes into an ⁇ -helical coiled-coil (compare FIG. 3A and FIG. 1 ) is not a trivial task of protein engineering. In this invention it is demonstrated how these peptide sequences that have an extended conformation when bound to the respective MHC molecule can nevertheless be incorporated into an ⁇ -helical coiled-coil oligomerization domain.
  • Optional substituents of D1, D2 and L are e.g. B-cell epitopes, targeting entities, or substituents reinforcing the adjuvant properties of the nanoparticle, such as an immunostimulatory nucleic acid, preferably an oligodeoxynucleotide containing deoxyinosine, an oligodeoxynucleotide containing deoxyuridine, an oligodeoxynucleotide containing a CG motif, or an inosine and cytidine containing nucleic acid molecule.
  • an immunostimulatory nucleic acid preferably an oligodeoxynucleotide containing deoxyinosine, an oligodeoxynucleotide containing deoxyuridine, an oligodeoxynucleotide containing a CG motif, or an inosine and cytidine containing nucleic acid molecule.
  • substituents reinforcing the adjuvant properties of the nanoparticle are antimicrobial peptides, such as cationic peptides, which are a class of immunostimulatory, positively charged molecules that are able to facilitate and/or improve adaptive immune responses.
  • An example of such a peptide with immunopotentiating properties is the positively charged artificial antimicrobial peptide KLKLLLLLKLK (SEQ ID NO: 63) which induces potent protein-specific type-2 driven adaptive immunity after prime-boost immunizations.
  • a particular targeting entity considered as substituent is an ER-targeting signal, i.e. a signal peptide that induces the transport of a protein or peptide to the endoplasmic reticulum (ER).
  • acyl group e.g. acetyl
  • a free amino group in particular to the N-terminal amino acid, or amino bound to the free carboxy group of the C-terminal amino acid to give a carboxamide function.
  • Optional substituents e.g. those optional substituents described hereinabove, are preferably connected to suitable amino acids close to the free end of the oligomerization domain D1 and/or D2. On self-assembly of the peptide nanoparticle, such substituents will then be presented at the surface of the SAPN.
  • the substituent is another peptide sequence S1 and/or S2 representing a simple extension of the peptide chain D1-L-D2 at either end or at both ends to generate a combined single peptide sequence of any of the forms S1-D1-L-D2, D1-L-D2-S2, or S1-D1-L-D2-S2, wherein S1 and S2 are peptidic substituents as defined hereinbefore and hereinafter.
  • the substituents S1 and/or S2 are said to extend the core sequence D1-L-D2 of the SAPN. Any such peptide sequence S1-D1-L-D2, D1-L-D2-S2, or S1-D1-L-D2-S2 may be expressed in a recombinant protein expression system as one single molecule.
  • a preferred substituent S1 and/or S2 is a B-cell epitope.
  • Other B-cell epitopes considered are hapten molecules such as a carbohydrate or nicotine, which are likewise attached to the end of the oligomerization domains D1 and/or D2, and hence will be displayed at the surface of the SAPN.
  • a tendency to form oligomers means that such peptides can form oligomers depending on the conditions, e.g. under denaturing conditions they are monomers, while under physiological conditions they may form, for example, trimers. Under predefined conditions they adopt one single oligomerization state, which is needed for nanoparticle formation. However, their oligomerization state may be changed upon changing conditions, e.g. from dimers to trimers upon increasing salt concentration (Burkhard P. et al., Protein Science 2000, 9:2294-2301) or from pentamers to monomers upon decreasing pH.
  • a building block architecture according to formula (I) is clearly distinct from viral capsid proteins.
  • Viral capsids are composed of either one single protein, which forms oligomers of 60 or a multiple thereof, as e.g. the hepatitis virus B particles (EP 1 262 555, EP 0 201 416), or of more than one protein, which co-assemble to form the viral capsid structure, which can adopt also other geometries apart from icosahedra, depending on the type of virus (Fender P. et al., Nature Biotechnology 1997, 15:52-56).
  • SAPN Self-assembling peptide nanoparticles
  • Peptidic oligomerization domains are well-known (Burkhard P. et al., Trends Cell Biol 2001, 11:82-88). The most simple oligomerization domain is probably the coiled-coil folding motif. This oligomerization motif has been shown to exist as a dimer, trimer, tetramer and pentamer. Some examples are the GCN4 leucine zipper, fibritin, tetrabrachion and COMP, representing dimeric, trimeric, tetrameric and pentameric coiled coils, respectively.
  • a coiled-coil is a peptide sequence with a contiguous pattern of mainly hydrophobic residues spaced 3 and 4 residues apart, usually in a sequence of seven amino acids (heptad repeat) or eleven amino acids (undecad repeat), which assembles (folds) to form a multimeric bundle of helices. Coiled-coils with sequences including some irregular distribution of the 3 and 4 residues spacing are also contemplated. Hydrophobic residues are in particular the hydrophobic amino acids Val, Ile, Leu, Met, Tyr, Phe and Trp. Mainly hydrophobic means that at least 50% of the residues must be selected from the mentioned hydrophobic amino acids.
  • D1 and/or D2 is a peptide of any of the formulae [aa(a)-aa(b)-aa(c)-aa(d)-aa(e)-aa(f)-aa(g)] X (IIa), [aa(b)-aa(c)-aa(d)-aa(e)-aa(f)-aa(g)-aa(a)] X (IIb), [aa(c)-aa(d)-aa(e)-aa(f)-aa(g)-aa(b)] X (IIc), [aa(d)-aa(e)-aa(f)-aa(g)-aa(b)-aa(c)] X (IId), [aa(e)-aa(f)-aa(g)-aa(b)-aa(c)] X (IId), [aa(e)-aa
  • Hydrophobic amino acids are Val, Ile, Leu, Met, Tyr, Phe and Trp.
  • a heptad is a heptapeptide of the formula aa(a)-aa(b)-aa(c)-aa(d)-aa(e)-aa(f)-aa(g) (IIa) or any of its permutations of formulae (IIb) to (IIg).
  • a charged amino acid able to form an inter-helical salt bridge to an amino acid of a neighboring heptad is, for example, Asp or Glu if the other
  • aa(a) is selected from Val, Ile, Leu and Met, and a derivative thereof, and
  • aa(d) is selected from Leu, Met and Ile, and a derivative thereof.
  • At least one aa(g) is selected from Asp and Glu and aa(e) in a following heptad is Lys, Arg or His; and/or
  • At least one aa(g) is selected from Lys, Arg and His, and aa(e) in a following heptad is Asp or Glu, and/or
  • At least one aa(a to g) is selected from Lys, Arg and His, and an aa(a to g) 3 or 4 amino acids apart in the sequence is Asp or Glu.
  • Such pairs of amino acids aa(a to g) are, for example aa(b) and aa(e) or aa(f).
  • D1 and/or D2 is a peptide that contains at least a sequence two heptad-repeats long that is predicted by the coiled-coil prediction program COILS to form a coiled-coil with higher probability than 0.9 for all its amino acids with at least one of the window sizes of 14, 21, or 28.
  • D1 and/or D2 is a peptide that contains at least one sequence three heptad-repeats long that is predicted by the coiled-coil prediction program COILS to form a coiled-coil with higher probability than 0.9 for all its amino acids with at least one of the window sizes of 14, 21, or 28.
  • D1 and/or D2 is a peptide that contains at least two separate sequences two heptad-repeats long that are predicted by the coiled-coil prediction program COILS to form a coiled-coil with higher probability than 0.9 for all its amino acids with at least one of the window sizes of 14, 21, or 28.
  • This pentamerization domain has the sequence LAPQMLRELQETNAALQDVRELLRQQVKQITFLKNTVMECDACG (SEQ ID NO:1). Small modifications of this domain are also envisaged. Such modifications may be e.g.
  • Modification of amino acids affecting the oligomerization state of this domain are also envisaged, resulting e.g. in a transition from pentamer to tetramer.
  • modifications of surface amino acids of this domain may include substitutions of amino acids (e.g. by cysteine or lysine) for the generation of attachment sites for functional groups.
  • This pentamerization domain has the sequence SSNAKWDQWSSDWQTWNAKWDQWSNDWNAWRSDWQAWKDD WARWNQRWDNWAT (SEQ ID NO:2). Small modifications of this domain are also envisaged.
  • Such modifications may be, e.g., the substitution of amino acids at the outside of the pentamer at positions aa(b), aa(c) or aa(f), preferably in position aa(f), by Cys for the purpose of the formation of a disulfide bridge between adjacent domains.
  • Other modifications of surface amino acids of this domain may include substitutions of amino acids for optimizing the interactions at the interface between adjacent oligomerization domains such as hydrophobic, hydrophilic or ionic interactions, or covalent bonds such as disulfide bridges. Also shorter constructs of this domain are envisaged.
  • Modification of amino acids affecting the oligomerization state of this domain are also envisaged, resulting, for example, in a transition from pentamerization domain to tetramerization domain exchanging core residues Trp by Phe.
  • Other core residue mutations as in Example 10 are also considered, but at least 70% of the core positions aa(a) and aa(d) have to be either a Trp or another aromatic amino acid.
  • Yet other modifications of surface amino acids of this domain may include substitutions of amino acids (e.g. by cysteine or lysine) for the generation of attachment sites for functional groups.
  • This tetramerization domain has the sequence IINETADDIVYRLTVIIDDRYESLKNLITLRADRL MIINDNVSTILASG (SEQ ID NO:64).
  • the sequences of coiled coils are characterized by a heptad repeat of seven residues with a 3,4-hydrophobic repeat.
  • the next periodicity that allows residues to assume quasi-equivalent positions after a small number of turns is three turns or 11 residues.
  • the C-terminus of the surface layer glycoprotein tetrabrachion from the hyperthermophilic archae-bacterium Staphylothermus marinus forms a right-handed coiled coil structure. It forms a tetrameric ⁇ -helical coiled coil stalk 70 nm long that is anchored to the cell membrane at its C-terminal end.
  • This tetrameric coiled-coil contains a series of HTL epitopes (Example 9) and hence is ideally suited as core oligomer of the self-assembling peptide nanoparticle (SAPN).
  • one oligomerization domain D1 or D2 is the trimerization domain (foldon) of the bacteriophage T4 protein fibritin (Tao, Y. et al., Structure 1997, 5:789-798) or a derivative thereof.
  • modifications of surface amino acids of this domain may include substitutions of residues for optimizing the interactions at the interface between adjacent oligomerization domains such as hydrophobic, hydrophilic or ionic interactions or covalent bonds like disulfide bridges.
  • modifications of surface amino acids of this domain may include substitutions of amino acids (e.g. by cysteine or lysine) for the generation of attachment sites for functional groups.
  • SAPN Self-assembling peptide nanoparticles
  • monomeric building blocks of formula (I) If such building blocks assemble, they will form so-called “even units”.
  • the number of monomeric building blocks, which will assemble into such an even unit will be defined by the least common multiple (LCM).
  • LCM least common multiple
  • the linker segment L has the appropriate length, this even unit may assemble in the form of a spherical peptidic nanoparticle.
  • the number of monomers needed to form an even unit will be 12 ( FIG. 2B ).
  • the least common multiple is always larger than m and n.
  • SAPN Self-assembling peptide nanoparticles
  • Table 2 Such SAPN represent topologically closed structures.
  • the tetrahedron has a 2-fold and two 3-fold axes
  • the cube and the octahedron have a 2-fold, a 3-fold and a 4-fold rotational symmetry axis
  • the dodecahedron and the icosahedron have a 2-fold, a 3-fold and a 5-fold rotational symmetry axis.
  • the spatial orientation of these axes is exactly the same as in the octahedron, and also in the dodecahedron and the icosahedron the spatial orientation of these axes relative to each other is exactly the same.
  • the cube and the octahedron, and similarly the dodecahedron and the icosahedron can be considered to be identical.
  • the cube/octahedron is built up from 24 identical three-dimensional building blocks, while the dodecahedron/icosahedron is built up from 60 identical three-dimensional building blocks (Table 2). These building blocks are the asymmetric units (AUs) of the polyhedron.
  • each of the pyramid edges corresponds to one of the rotational symmetry axes, hence these AUs will carry at their edges 2-fold, 3-fold, and 4-fold or 5-fold symmetry elements depending on the polyhedron type.
  • these symmetry elements are generated from peptidic oligomerization domains such AUs are constructed from monomeric building blocks as described above. It is sufficient to align the two oligomerization domains D1 and D2 along two of the symmetry axes of the AU ( FIG. 3 ). If these two oligomerization domains form stable oligomers, the symmetry interface along the third symmetry axis will be generated automatically, and it may be stabilized by optimizing interactions along this interface, e.g. hydrophobic, hydrophilic or ionic interactions, or covalent bonds such as disulfide bridges.
  • SAPN self-assembling peptide nanoparticles
  • a regular geometry dodecahedron, cube
  • more than one even unit is needed.
  • SAPN self-assembling peptide nanoparticles
  • 4 even units each composed of 15 monomeric building blocks are needed, i.e. the peptidic nanoparticle with regular geometry will be composed of 60 monomeric building blocks.
  • Table 2 The combinations of the oligomerization states of the two oligomerization domains needed and the number of even units to form any of the regular polyhedra are listed in Table 2.
  • the even units will further assemble to form regular polyhedra composed of more than one even unit depends on the geometrical alignment of the two oligomerizations domains D1 and D2 with respect to each other, especially on the angle between the rotational symmetry axes of the two oligomerization domains. This is governed by i) the interactions at the interface between neighboring domains in a nanoparticle, ii) the length of the linker segment L, iii) the shape of the individual oligomerization domains. This angle is larger in the even units compared to the arrangement in a regular polyhedron. Also this angle is not identical in monomeric building blocks as opposed to the regular polyhedron.
  • the angle between the two oligomerization domains is sufficiently small (even smaller than in a regular polyhedron with icosahedral symmetry), then a large number (several hundred) peptide chains can assemble into a peptidic nanoparticle. This can be achieved by replacing the two cysteine residues that are located at the interface between the two helices as in the original design of Raman S. et al., Nanomedicine: Nanotechnology, Biology, and Medicine 2006, 2:95-102, and that are forming a disulfide bridge between the two helices, by the small residue alanine as in sequence SEQ ID NO:33.
  • the angle between the two helices can get smaller and consequently more than 60 peptide chains can assemble into a SAPN. In such a design the SAPN have a molecular weight of about 4 MD, corresponding to about 330 peptide chains (Example 6).
  • the T-cell epitopes as opposed to the B-cell epitopes—do not need to be displayed on the surface of a carrier to cause immunization, they can be incorporated into the core scaffold of the SAPN, i.e. the coiled-coil sequence of an oligomerization domain.
  • the present invention it is shown how the features of MHC binding of T-cell epitopes, which requires an extended conformation for MHC binding ( FIG.
  • B-cell epitopes that are not coiled-coils are incorporated into the coiled-coil sequence of the SAPN oligomerization domain by inserting them between two stretches of coiled-coil segments, such that this whole sequence acts as a single oligomerization domain.
  • the coiled-coil scaffold can provide means to restrict the conformation of the B-cell epitope to a conformation that is nearly identical to its native conformation.
  • the T-cell epitopes can be chosen from different sources: For example, the T-cell epitopes can be determined by experimental methods, they are known from literature, they can be predicted by prediction algorithms based on existing protein sequences of a particular pathogen, or they may be de novo designed peptides or a combination of them.
  • SAPN self-assembling peptide nanoparticle
  • T-cell epitopes can be selected from a particular pathogen (e.g. as in Examples 12, 13 and 14), from a cancer specific peptide sequence (e.g. as in Example 4), or they may be de novo designed peptides with a particular feature, e.g. the PADRE peptide (U.S. Pat. No. 5,736,142) that binds to many different MHC II molecules, which makes it a so-called promiscuous T-cell epitope (e.g. as in Example 1).
  • MHC-database MHCBN VERSION 4.0
  • PDB-database Protein Data Bank
  • HTL epitopes It is well known and well documented that incorporation of HTL epitopes into an otherwise not immunogenic peptide sequence or attaching it to a non-peptidic antigen can make those much more immunogenic.
  • the PanDR binding peptide HTL epitope PADRE has widely been used in vaccine design for a malaria, Alzheimer and many others vaccines.
  • T-cell epitopes are peptides that have binding affinities (IC 50 values) of less than 50,000 nM to the corresponding MHC molecule. Such peptides are considered as MHC binders. According to this definition, as of August 2006, in the Version 4.0 of the MHCBN database the following data is available: 20717 MHC binders and 4022 MHC non-binders.
  • Suitable T-cell epitopes can also be obtained by using prediction algorithms. These prediction algorithms can either scan an existing protein sequence from a pathogen for putative T-cell epitopes, or they can predict, whether de novo designed peptides bind to a particular MHC molecule. Many such prediction algorithms are commonly accessible on the internet. Examples are SVRMHCdb (J. Wan et al., BMC Bioinformatics 2006, 7:463), SYFPEITHI, MHCPred, motif scanner or NetMHCIIpan for MHC II binding molecules and NetMHCpan for MHC I binding epitopes.
  • HTL epitopes as described herein and preferred for the design are peptide sequences that are either measured by biophysical methods or predicted by NetMHCIIpan to bind to any of the MHC II molecules with binding affinities (IC 50 values) better than 500 nM. These are considered weak binders. Preferentially these epitopes are measured by biophysical methods or predicted by NetMHCIIpan to bind to the MHC II molecules with IC 50 values better than 50 nM. These are considered strong binders.
  • CTL epitopes as described herein and preferred for the design are peptide sequences that are either measured by biophysical methods or predicted by NetMHCpan to bind to any of the MHC I molecules with binding affinities (IC 50 values) better than 500 nM. These are considered weak binders. Preferentially these epitopes are measured by biophysical methods or predicted by NetMHCpan to bind to the MHC I molecules with IC 50 values better than 50 nM. These are considered strong binders.
  • the T-cell epitopes can be incorporated at several places within the peptide sequence of the coiled-coil oligomerization domains D1 and or D2. To achieve this, the particular sequence with the T-cell epitope has to obey the rules for coiled-coil formation as well as the rules for MHC binding.
  • the rules for coiled-coil formation have been outlined in detail above.
  • the rules for binding to MHC molecules are incorporated into the MHC binding prediction programs that use sophisticated algorithms to predict MHC binding peptides.
  • binding motifs are summarized in Table 3.
  • the motif shows x for positions that can have any amino acid, and in square brackets the (list of) amino acids that can only be at a particular position of the binding motif.
  • the motif x-[VTILF]-x-x-x-x-x-x-[YF(ML)] means that second and C-terminal positions are anchor positions.
  • the dominant amino acids at the second position are V, T, I, L, F, and at the C-terminal anchor position the dominant amino acids are Y and F, while M and L are preferred but not dominant.
  • Marsh2000 Marsh S. G. E., Parham P. and Barber L. D., The HLA Factsbook. Academic Press, San Diego, 2000. URL: http://www.anthonynolan.com/HIG/.
  • SYFPEITHI The SYFPEITHI Database of MHC Ligands, Peptide Motifs and Epitope Prediction. January 2003. URL: http://www.syfpeithi.de. Luscher2001: Luscher M. A. et al., Immunogenetics. 2001, 53(1): 10-14. Yusim2004: Yusim K. et al., Appl Bioinformatics 2005, 4(4): 217-225.
  • HLA supertypes Many of the MHC molecules have very similar binding motifs and hence these can be grouped into so-called HLA supertypes.
  • the binding motifs for these supertypes are summarized in Table 4.
  • the frequency of occurrence of a particular amino acid at a certain position of the T-cell epitope can also be summarized.
  • positions 1, 4, 6 and 9 in a T-cell epitope are the most critical ones.
  • the most preferred residues at these positions are listed in Table 5, however, the preferences for particular amino acids at these position vary largely between the different MHC molecules. Therefore, as mentioned above, binding of a particular amino acid sequence to a MHC molecule can be much more accurately predicted by the prediction programs listed above.
  • a peptide sequence obeys both, T-cell binding motif restriction as well as coiled-coil heptad repeat motif restriction, it can be incorporated into the coiled-coil oligomerization domain of the SAPN. This can be achieved for a large number of T-cell epitopes by adjusting the alignment of peptide sequence such that the T-cell binding motif overlaps with the coiled-coil forming motif.
  • a candidate T-cell epitope has to be chosen by using known T-cell epitopes from the literature or from databases or predicted T-cell epitopes by using a suitable epitope prediction program.
  • a proteasomal cleavage site has to be inserted at the C-terminal end of the CTL epitopes. This can be done by using the prediction program for proteasomal cleavage sites PAProc (Hadeler K. P. et al., Math. Biosci.
  • the sequence of the T-cell epitope has to be aligned with the coiled-coil sequence such that it is best compatible with the rules for coiled-coil formation as outlined above. Whether the sequence with the incorporated T-cell epitope will indeed form a coiled-coil can be predicted, and the best alignment between the sequence of the T-cell epitope and the sequence of the coiled-coil repeat can be optimized by using coiled-coil prediction programs such as COILS (Gruber M. et al., J. Struct. Biol. 2006, 155(2):140-5) or MULTICOIL which are available on the internet.
  • COILS Garnier M. et al., J. Struct. Biol. 2006, 155(2):140-5
  • MULTICOIL which are available on the internet.
  • the T-cell epitope may be incorporated into the oligomerization domain (see Example 3).
  • the T-cell epitope has to be flanked by strong coiled-coil forming sequences of the same oligomerization state. This will either stabilize the coiled-coil structure to a sufficient extent or alternatively it can generate a loop structure within this coiled-coil oligomerization domain. This is essentially the same procedure as described in the next section for the incorporation of B-cell epitopes into the coiled-coil core sequence of the SAPN.
  • the incorporation into the coiled-coil core of the SAPN of small B-cell epitopes that are not ⁇ -helical is envisaged. This can be accomplished by the same procedure as outlined above for the T-cell epitopes that are not compatible with a coiled-coil structure.
  • the structure of T4 fibritin (pdb accession code 1aa0) contains two loops structures within its coiled-coil. The loops protrude from the coiled-coil helix between two helical turns such that the helical structure of the coiled-coil is not interrupted.
  • the loops leave the helix at position aa(b) of the coiled-coil and reenter the helix at position aa(c) of the coiled-coil sequence.
  • One of the loops is a short beta-turn while the other is a more irregular loop structure.
  • the spacing between the residues aa(b) and aa(c) in a coiled-coil is ideally suited to serve as anchor points for an antiparallel beta-turn peptide.
  • residues aa(b) or aa(c) or both of them are glycine residues this allows for the needed flexibility of the protein secondary structure to exit and reenter the alpha-helix of the coiled-coil.
  • CTL epitopes require a proteasomal cleavage site at their C-terminal end.
  • the epitopes should not be similar to human sequences to avoid autoimmune responses—except when it is the goal to elicit an immune response against a human peptide. Possible examples are the cancer-specific CTL epitopes of Example 4.
  • a SAPN is preferred wherein at least one of the T-cell epitopes is a CTL epitope, and, in particular, wherein the sequence further contains a proteasomal cleavage site after the CTL epitope.
  • a SAPN wherein at least one of the T-cell epitopes is a HTL epitope, in particular, a pan-DR-binding HTL epitope.
  • HTL epitope in particular, a pan-DR-binding HTL epitope.
  • pan-DR-binding HTL epitopes bind to many of the MHC class II molecules as listed at the bottom of Table 3 and are therefore recognized in a majority of healthy individuals, which is critical for a good vaccine.
  • a SAPN wherein the sequence D1-L-D2 contains a series of overlapping T-cell epitopes, either if D1 or D2 are a trimer (Examples 7 and 8), a tetramer (Examples 9) or a pentamer (Examples 10).
  • B-cell epitopes need to be displayed at the surface of the SAPN. They may or may not be part of the coiled-coil sequence, i.e. the coiled-coil itself may partially be a B-cell epitope depending on whether the portion of the coiled-coil is surface accessible.
  • the B-cell epitope composed of the trimeric coiled-coil of the surface proteins of enveloped viruses can be displayed on the surface of the SAPN and be part of coiled-coil sequence at the same time.
  • An example of such a design is presented in Raman S. et al., Nanomedicine: Nanotechnology, Biology, and Medicine 2006; 2:95-102.
  • Coiled-coils of any oligomerization state in general are exceptionally well-suited to be presented in conformation specific manner by the SAPN.
  • Coiled-coils are abundant, not only in enveloped virus surface proteins but also, for example, in the genome of the malaria pathogen Plasmodium falciparum (Villard V. et al., PLoS ONE 2007; 2(7):e645).
  • the B-cell epitopes will not be part of the coiled-coil oligomerization domains, or they may be composed of a coiled-coil and an additional portion that is not a coiled-coil, as for example the trimeric autotransporter adhesions (TAA) of bacteria, which have a coiled-coil stalk and a globular head domain, such as the TAA of N. meningitidis.
  • TAA trimeric autotransporter adhesions
  • proteins as B-cell epitopes that are themselves oligomeric, such as trimeric hemagglutinin, and the tetrameric sialidase or M2 surface proteins of influenza.
  • Such a vaccine preferably contains all three types of epitopes, B-cell, HTL and CTL epitopes.
  • B-cell Preferably only one (or very few) B-cell epitope should be placed at either end of the peptide chains. This will place the B-cell epitope on the surface of the SAPN in a repetitive antigen display.
  • the HTL epitopes should be as promiscuous as possible. They do not necessarily need to be derived from the pathogen but can be peptides that elicit a strong T-help immune response. An example would be the PADRE peptide. Preferably these are the T-cell epitopes that are incorporated into the D1-L-D2 core sequence of the SAPN.
  • the CTL epitopes need to be pathogen specific, they need to have C-terminal proteasomal cleavage sites. Since the T-cell epitopes do not require repetitive antigen display several different T-cell epitopes can be incorporated into one single SAPN by co-assembly of different peptide chains that all have the same nanoparticle forming D1-L-D2 core but carry different T-cell epitopes that are not part of the core forming sequence and hence would not be incorporated into the coiled-coil sequences.
  • peptide chains carrying an ER-targeting signal i.e. a signal peptide that induces the transport of a protein or peptide to the endoplasmic reticulum (ER) can be co-assembled into the same SAPN to bring the CTL epitopes into the ER for proper presentation by the MHC I molecules since cross-presentation is not very efficient in humans.
  • the ER targeting signal does not need to be on a separate peptide chain, it can be in the same peptide as the CTL epitopes.
  • a suitable ER signal peptide would be for example the ER targeting signal (E3/19K) MRYMILGLLALAAVCSA (SEQ ID NO:6).
  • a therapeutic vaccine aimed to generate a strong antibody response is particularly useful for the treatment of Alzheimer, hypertension, obesity, drug addictions, or inflammation.
  • a vaccine preferably only one B-cell epitope is used.
  • the strong humoral immune response due to repetitive antigen display can be further enhanced by including one or more promiscuous HTL epitopes into the SAPN.
  • B-cell epitope no B-cell epitope has to be used.
  • the immune response against the particular CTL epitopes (for example MAGE-1,2,3; MART-1,2,3; or Her-2/neu, see also Example 4) is further enhanced by including one or more promiscuous HTL epitopes into the SAPN.
  • SAPN Self-Assembling Peptide Nanoparticles
  • a SAPN that is composed of many HTL epitopes will induce a strong T-help immune response (see Example 2). If given in the same dose with any other vaccine formulation this will result in a stimulation of the immune response.
  • Such a SAPN will be an adjuvant without the need of any CTL or B-cell epitope.
  • B-cell and CTL epitopes can be combined with such an adjuvant SAPN.
  • adjuvant molecules can be covalently coupled to the SAPN as a substituent to the oligomerization domain D1 or D2 to further stimulate the adjuvant effect of the SAPN.
  • immunostimulatory nucleic acids preferably an oligodeoxynucleotide containing deoxyinosine, an oligodeoxynucleotide containing deoxyuridine, an oligodeoxynucleotide containing a CG motif, an inosine and cytidine containing nucleic acid molecule.
  • Other immunostimulatory molecules are, for example, antimicrobial peptides such as cationic peptides which are a class of immunostimulatory, positively charged molecules that are able to facilitate and/or improve adaptive immune responses.
  • peptide with immunopotentiating properties is the positively charged artificial antimicrobial peptide KLKLLLLLKLK (SEQ ID NO: 63), which induces potent protein-specific type-2 driven adaptive immunity after prime-boost immunizations.
  • antigens of the invention are selected from the group consisting (a) proteins suited to induce an immune response against cancer cells; (b) proteins or carbohydrates suited to induce an immune response against infectious diseases; (c) proteins suited to induce an immune response against allergens; (d) peptide hormones suited to induce an immune response for the treatment of a human disease; and (e) hapten molecules suited to induce an immune response to treat addictions or other disorders.
  • Peptidic nanoparticles comprising such proteins, peptidic fragments thereof, peptides, carbohydrates, or haptens may be suited to induce an immune response in humans, or also in farm animals and pets.
  • the antigens or antigenic determinants are the ones that are useful for the prevention of infectious disease.
  • Such treatment will be useful to prevent a wide variety of infectious diseases affecting a wide range of hosts, e.g. humans or non-human animals, such as cow, sheep, pig, dog, cat, other mammalian species and non-mammalian species as well.
  • SAPN comprising one of the following antigens:
  • Treatable infectious diseases are well known to those skilled in the art. Examples include infections of viral, bacterial or parasitic etiology such as the following diseases: Amoebiasis, Anthrax, Campylobacter infections, Chickenpox, Cholera, Dengue, Diphtheria, Encephalitis, Ebola, Influenza, Japanese Encephalitis, Leishmaniasis, Malaria, Measles, Meningococcal Disease, Mumps, Nosocomial infections, Pertussis, Pneumococcal Disease, Polio (Poliomyelitis), Rubella, Shingles, Shistosomiasis, Tetanus, Tick-Borne Encephalitis, Trichomoniasis, Trypanosomiasis, Tuberculosis, Typhoid, Varicella, and Yellow Fever.
  • viruses include infections of viral, bacterial or parasitic etiology such as the following diseases: Amoebiasis, Anthrax, Campylobacter
  • SAPN comprising one of the following antigens from the following parasites:
  • Campylobacter Cytomegalovirus, Epstein-Barr Virus, FMDV, Haemophilus influenzae Type b, Helicobacter pylori , Hepatitis B Virus, Hepatitis C Virus, Hepatitis E Virus, Herpes Simplex Virus, Human Immunodeficiency Virus, Human Papillomavirus, Neisseria meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae , Respiratory Syncytial Virus, Rotavirus, Roundworm, Hookworm, and West Nile Virus.
  • a composition for the prevention and treatment of malaria is envisaged (Example 11).
  • the life cycle of the malaria parasite provides several stages at which interference could lead to cessation of the infective process.
  • a human becomes infected with malaria from the bite of a female Anopheles mosquito.
  • the mosquito inserts its probe into a host and in so doing, injects a sporozoite form of Plasmodium falciparum (or vivax), present in the saliva of the mosquito.
  • Possible protein and peptide sequences suitable for the design of a peptide vaccine may contain sequences from the following Plasmodium proteins: MSP-1 (a large polymorphic protein expressed on the parasite cell surface), MSA1 (major merozoite surface antigen 1), CS protein (native circumsporozoite), 35 KD protein or 55 KD protein or 195 KD protein according to U.S. Pat. No. 4,735,799, AMA-1 (apical membrane antigen 1), or LSA (liver stage antigen).
  • MSP-1 a large polymorphic protein expressed on the parasite cell surface
  • MSA1 major merozoite surface antigen 1
  • CS protein native circumsporozoite
  • 35 KD protein or 55 KD protein or 195 KD protein according to U.S. Pat. No. 4,735,799 AMA-1 (apical membrane antigen 1)
  • LSA liver stage antigen
  • one of the B-cell epitopes is a sequence of 8 to about 48 residues that constitute a B cell epitope of the circumsporozoite (CS) protein.
  • This B-cell epitope is a redundant repeat region of the amino acid sequence NANP for Plasmodium falciparum .
  • this B cell epitope comprises two to about five repeats of the amino acid residue sequence NANP or the permutations thereof ANPN, NPNA, and PNAN.
  • the corresponding repeat region in Plasmodium vivax is composed of any of the following highly similar sequences
  • one of the B-cell epitopes is a sequence of 8 to about 48 residues composed of any of these sequences.
  • Table 7 lists preferred P. falciparum coiled-coil B-cell epitopes (Villard V. et al., PLoS ONE 2007, 2(7):e645 and Agak G. W., Vaccine (2008) 26, 1963-1971. Since for B-cell epitopes only the surface accessible residues are of critical importance for their interactions with the B-cell receptor and the production of antibodies, the coiled-coil core residues at aa(a) and aa(d) positions, which are not surface exposed can be modified to some extent without changing the ability of the immunogen to elicit neutralizing antibodies.
  • the coiled-coil B-cell epitope with modifications of one or more of their core positions is a peptide that contains at least a sequence which is two heptad-repeats long that is predicted by the coiled-coil prediction program COILS to form a coiled-coil with higher probability than 0.9 for all its amino acids with at least one of the window sizes of 14, 21, or 28.
  • the coiled-coil B-cell epitope with modifications of one or more of their core positions is a peptide that contains at least one sequence three heptad-repeats long that is predicted by the coiled-coil prediction program COILS to form a coiled-coil with higher probability than 0.9 for all its amino acids with at least one of the window sizes of 14, 21, or 28.
  • the coiled-coil B-cell epitope with modifications of one or more of their core positions is a peptide that contains at least two separate sequences two heptad-repeats long that are predicted by the coiled-coil prediction program COILS to form a coiled-coil with higher probability than 0.9 for all its amino acids with at least one of the window sizes of 14, 21, or 28.
  • B-cell epitope sequences B-cell epitope SEQ ID NO: IKTMNTQISTLKNDVHLLNEQIDKLNNEKGTLNSKISE 72 LNVQIMDL LLSKDKEIEEKNKKIKELNNDIKKL 73 ICSLTTEVMELNNKKNELIEENNKLNLVDQGKKKLKKD 74 VEKQKKEIEKL VDKIEEHILDYDEEINKSRSNLFQLKNEICSLTTEVME 75 LNNKKNELIEENNKLNLVDQGKKKLKKDVEKQKKEIEKL LDENEDNIKKMKSKIDDMEKEIKYR 76 GMNNMNGDINNIN(GDINNMN) 4 77 KKRNVEEELHSLRKNYNIINEEIEEIT 78 EEIKEEIKEVKEEIKEVKEEIKEVKEEIKEVKEEIKE 79 KNDINVQLD
  • a composition for the prevention and treatment of HIV is envisaged (Examples 5 and 12).
  • a synthetic peptide capable of eliciting HIV-specific antibodies may be used, said synthetic peptide having the amino acid sequence of a functional T-cell epitope or B-cell epitope of an envelope or gag protein or gp120 or gp41 of HIV-1 to provide an immune response.
  • sequences within gp41 or gp120 which can induce conformation specific neutralizing antibodies able to interfere with the fusion process like the known antibodies 2F5 and 4E10 or from the V3-loop of gp41 or gp120.
  • Such sequences are mainly localized in and around the HR1 and HR2 and the cluster I and cluster II regions.
  • Antibodies binding to e.g. the coiled-coil trimer of gp41 and elicited by peptidic nanoparticles of the invention incorporating this coiled-coil trimer will inhibit hairpin formation and hence viral fusion.
  • antibodies raised against the trimeric coiled-coil of Ebola or of another virus with a similar fusion process will inhibit viral entry of these viruses.
  • CTL epitopes were predicted using SVRMHCdb (Wan J. et al., BMC Bioinformatics 2006, 7:463). These conserved protein sequences contain CTL epitopes predicted to bind to the HLA molecules as listed in Table 10 and are preferred CTL epitopes for the design of an SAPN-HIV vaccine. These peptide epitopes contain largely overlapping sequences that can be combined to longer peptide sequences that harbor multiple CTL epitopes in one single continuous peptide string (Table 11).
  • HLA-restriction PLDEGFRKYTAFTIPSINNE 189 A*0101, A*0201, A*0202, A*0206, A*6802, B*3501, B*5301, A68 AVFIHNFKRKGGIGG 190 A*3101, A*1101, A11, A31, A68, B*5401, A*0204 IIGRNLLTQIGCTLNFPISPIETVPV 191 A*0207, B51, A2, B*0702, A*0204, A*0203 DFWEVQLGIPHPAGLKKKKSV 192 A*0201, B*0702, A*0301, A*0206, B*5401, A3 LTEEAELELAENREILKDPVHGV 193 A31, B35, B*3501, A*0204, A*0101 KGPAKLLWKGEGAV 194 A*0202, A*1101, A*0301,
  • Influenza A encodes an integral membrane protein, M2, a homotetramer, the subunit of which has a small external domain (M2e) of 23 amino acid residues.
  • M2e small external domain
  • the natural M2 protein is present in a few copies in the virus particle and hidden to the immune system by the bulky other two surface proteins hemagglutinin and sialidase. On the other hand it exists in abundance on the membrane surface of virus-infected cells.
  • the sequence of M2e is highly conserved.
  • the M2e tetramer is a highly conserved B-cell epitope for both, human and avian specific influenza strains (Table 12 and 13). In a preferred embodiment of this invention it can be displayed in its native tetrameric conformation when attached to the N-terminus of the tetrameric coiled-coil from tetrabrachion—or any other tetrameric coiled-coil—of the SAPN (Example 9).
  • Influenza hemagglutinin is activated by cleavage of the precursor protein into two separate peptide chains (Steinauer D. S. et al., Virology 1999; 258:1-20). Cleavage of the HA precursor molecule HA0 is required to activate virus infectivity, and the distribution of activating proteases in the host is one of the determinants of tropism and, as such, pathogenicity.
  • the HAs of mammalian and nonpathogenic avian viruses are cleaved extracellularly, which limits their spread in hosts to tissues where the appropriate proteases are encountered.
  • the HAs of pathogenic viruses are cleaved intracellularly by ubiquitously occurring proteases and therefore have the capacity to infect various cell types and cause systemic infections.
  • the N-terminal part of the cleavage peptide is not highly conserved (the C-terminal portion of the cleavage peptide is in fact highly conserved).
  • the cleavage peptide is surface exposed and the six residues (three residues on each side of the cleavage site) around the cleavage site are the most characteristic of this peptide sequence (highlighted in bold in Table 14).
  • these six residues represent the B-cell epitope, which can induce antibodies that, upon binding to the peptide, can protect the HA precursor protein from getting cleaved.
  • the SAPN are ideally suited to display a multitude of different cleavage sequences specific for the different HA types (Table 14) by co-assembling peptides that have the same SAPN-forming core D1-L-D2 but different B-cell epitopes attached to it (Example 15).
  • hemagglutinin B-cell epitope string comprising the sequences of H1, H2, and H3 cleavage sites with an inserted aspartate amino acid to make the sequence more soluble and less basic would look like this: SIQSRGLFGDIESRGLFGERQTRGIFG (SEQ ID NO:227).
  • Peptides with the same core sequence but different B-cell epitopes or epitope strings can be co-assembled into a single SAPN to generate a multivalent SAPN immunogen that possibly includes all or the most important (H1, H2, H3, H5, H7 and H9 for a human vaccine) sequences of Table 14 (Example 15).
  • an Influenza vaccine SAPN composed of six peptide chains with an identical core and identical N-terminal B-cell epitope M2e and about 20 CTL epitopes at the C-terminus (three or four each per peptide chain) can be co-assembled into a single SAPN (Example 14).
  • Table 15 preferred conserved CTL epitopes from Parida R. et al., Vaccine 2007, 25:7530-7539 are listed. Since the core of these six peptide chains is identical, co-assembly of these six peptide chains into one single SAPN allows the incorporation of about 20 different CTL epitopes into one single SAPN.
  • compositions of the invention are immunotherapeutics that may be used for the treatment of metabolic disorders and diseases, or addictions. Most preferred are immunotherapeutics for the treatment of Alzheimer disease, hypertension, obesity, nicotine- and cocaine addictions.
  • the A ⁇ -fragment (A ⁇ ) is a 42 amino acid long peptide (A ⁇ 1-42). Since the whole 42 residue long peptide sequence also contains CTL epitopes that can cause autoimmune reactions, it is desirable to use only shorter fragments of this peptide for vaccine design, such as A ⁇ 1-12 or even such short peptides as A ⁇ 1-6 (U.S. Pat. No. 7,279,165).
  • TNF ⁇ protein as an immunogen has some limitations. Local overproduction of the proinflammatory cytokine TNF ⁇ is critically involved in the pathogenesis of several chronic inflammatory disorders, including rheumatoid arthritis, psoriasis, and Crohn's disease. Neutralization of TNF ⁇ by monoclonal antibodies (mAbs, infliximab, adalimumab) or chimeric soluble receptors (etanercept) is efficacious in the treatment of these conditions but has several potential drawbacks. It may induce allotype- or Id-specific Abs, which might limit long-term efficacy in many patients.
  • mAbs, infliximab, adalimumab chimeric soluble receptors
  • TNF ⁇ -antagonists in particular mAbs
  • TNF ⁇ -antagonists increases the risk of opportunistic infections, especially those caused by intracellular pathogens like Mycobacterium tuberculosis, Listeria monocytogenes , or Histoplasma capsulatum .
  • Immunization with shorter fragments of TNF ⁇ comprising only residues 4-23 has been shown to avoid some of these problems (G. Spohn et al., The Journal of Immunology, 2007, 178: 7450-7457).
  • TNF ⁇ 4-23 immunization with TNF ⁇ 4-23 is a novel efficient therapy for rheumatoid arthritis and other autoimmune disorders, which adds a new level of safety to the existing anti-TNF ⁇ therapies.
  • TNF ⁇ 4-23 By selectively targeting only the soluble form of TNF ⁇ and sparing the transmembrane form, pathogenic effects of TNF ⁇ are neutralized by the vaccine, while important functions in the host response to intracellular pathogens remain intact.
  • opiates Apart from nicotine and cocaine also the following compounds may be used as hapten molecules the design of a B-cell SAPN vaccine for addictions: opiates, marijuana, amphetamines, barbiturates, glutethimide, methyprylon, chloral hydrate, methaqualone, benzodiazepines, LSD, anticholinergic drugs, antipsychotic drugs, tryptamine, other psychomimetic drugs, sedatives, phencyclidine, psilocybine, volatile nitrite, and other drugs inducing physical dependence and/or psychological dependence.
  • compositions of the invention are immuno-therapeutics that may be used for the treatment of allergies.
  • the selection of antigens or antigenic determinants for the composition and the method of treatment for allergies would be known to those skilled in the medical art treating such disorders.
  • Representative examples of this type of antigen or antigenic determinant include bee venom phospholipase A2, Bet v I (birch pollen allergen), 5 Dol m V (white-faced hornet venom allergen), and Der p I (House dust mite allergen).
  • compositions of the invention are immuno-therapeutics that may be used for the treatment of cancer.
  • Major cancers for targeted vaccines design are the following: Brain Cancer, Breast Cancer, Cervical Cancer, Colorectal Cancer, Esophageal Cancer, Glioblastoma, Leukemia (acute Myelogenous and chronic Myeloid), Liver cancer, Lung Cancer (Non-Small-Cell Lung Cancer, Small-Cell Lung Cancer), Lymphoma (Non-Hodgkin's Lymphoma), Melanoma, Ovarian Cancer, Pancreatic Cancer, Prostate Cancer, Renal Cancer.
  • antigens or antigenic determinants for the composition and method of treatment for cancer would be known to those skilled in the medical art treating such disorders.
  • Representative examples of this type of antigen or antigenic determinant include the following: HER2/neu (breast cancer), GD2 (neuroblastoma), EGF-R (malignant glioblastoma), CEA (medullary thyroid cancer), CD52 (leukemia), MUC1 (expressed in hematological malignancies), gp100 protein, or the product of the tumor suppressor gene WT1.
  • HER2/neu breast cancer
  • GD2 neuroblastoma
  • EGF-R malignant glioblastoma
  • CEA medullary thyroid cancer
  • CD52 leukemia
  • MUC1 expressed in hematological malignancies
  • gp100 protein or the product of the tumor suppressor gene WT1.
  • Table 17 cancer specific T-cell epitopes of interest are shown with the relevant protein of origin and the MHC restriction.
  • PADRE P5c-8-Mal
  • the pan-DR epitope PADRE with the sequence AKFVAAWTLKAAA (SEQ ID NO:296) is incorporated into the trimeric coiled-coil of the SAPN by using the following design criteria:
  • the residue alanine (A) has a strong tendency to form alpha-helices.
  • the alignment with coiled-coil core positions is such that the residues valine, tryptophan and alanine are at an aa(a), aa(d) and again an aa(a) position of the heptad repeat pattern.
  • the remaining part of the trimeric coiled-coil N-terminal and C-terminal to the HTL epitope is designed such that the sequence is predicted to from a very strong coiled-coil.
  • the sequence of this peptide (SEQ ID NO:7) is predicted to form a coiled-coil by the prediction program COILS.
  • the coiled-coil forming probability is more than 95% for all the residues in the sequence using a window of 21 amino acids for the coiled-coil prediction (see Table 18 below). Hence this is a predicted coiled-coil that also contains a T-cell epitope.
  • the coiled-coil prediction drops to very low values of less than 2% probability within the sequence of the T-cell epitope.
  • the larger window size of 21 amino acids with its high predicted coiled-coil propensities throughout the sequence shows the effect of the flanking regions at the N- and C-terminal ends of the T-cell epitope. If these are sequences that strongly favor coiled-coil formation then the less favorable coiled-coil propensity of the T-cell epitope may be compensated and the whole sequence will be induced to form a coiled-coil even though the T-cell epitope does not contain a very favorable coiled-coil sequence.
  • This trimeric coiled-coil of SEQ ID NO:7 was then included in the SAPN sequence of SEQ ID NO:8 below, which is composed of a His-tag, the pentameric coiled-coil of COMP, the trimeric coiled-coil composed of the sequence of SEQ ID NO:7 comprising the PADRE T-cell epitope, and the B-cell epitope from the CS-protein of Plasmodium berghei .
  • the peptide with this sequence was expressed in E. coli and purified on a nickel affinity column by standard biotechnology procedures.
  • the refolding was performed according to Raman S. et al., Nanomedicine: Nanotechnology, Biology, and Medicine 2 (2006) 95-102.
  • the refolded SAPN were analyzed for nanoparticle formation by dynamic light scattering (DLS) techniques and transmission electron microscopy (TEM).
  • the DLS analysis showed a nice size distribution with an average particle diameter of 32.01 nm and polydispersity index of 12.9% ( FIG. 4A ).
  • the TEM pictures ( FIG. 4B ) show nanoparticles of the same size as determined by DLS.
  • This coiled-coil sequence contains four overlapping HTL epitopes with the sequences LEELERSIW (residues 6-14 of SEQ ID NO:9), IWMLQQAAA (residues 13-21 of SEQ ID NO:8), WMLQQAAR (residues 14-22 of SEQ ID NO:9), and MLQQAAARL (residues 15-23 of SEQ ID NO:9) that are predicted by the algorithm SVRMHC to bind to the different MHC II molecules DQA1*0501, DRB1*0501, DRB5*0101, and DRB1*0401 respectively, with predicted binding affinities (pIC50 values) of 6.122, 8.067, 6.682, and 6.950 respectively.
  • HTL epitopes are aligned with the coiled-coil heptad repeat such that they are predicted to form a very strong coiled-coil.
  • the aa(a) and aa(d) core positions of the coiled-coil are occupied by Leu, Leu, Ile, Leu, Ala and Leu, most of them very good residues for high coiled-coil forming propensity with only Ala being somewhat less favorable.
  • the sequence of the peptide is predicted to form a coiled-coil by the prediction program COILS.
  • the coiled-coil forming probability is more than 99% for all the residues of the HTL epitopes in the sequence (Table 19). Comparing the small and large window sizes for coiled-coil prediction shows again the influence of the flanking sequences for the coiled-coil stability. With a window size of 28 amino acids the whole sequence is predicted to form a coiled-coil with a probability of 100% while the smaller window size of 14 amino acids shows the effect of the lower coiled-coil propensity of the T-cell epitopes at the N-terminal end with lower prediction values for coiled-coil formation. Hence for the whole sequence this peptide is predicted to from a stable coiled-coil including the four different HTL epitopes.
  • This trimeric coiled-coil was then included into the SAPN sequence SEQ ID NO:10 below, composed of a His-tag, the pentameric coiled-coil of COMP, the trimeric coiled-coil of SEQ ID NO:9 and the B-cell epitope from the CS-protein of Plasmodium berghei .
  • the peptide with this sequence was expressed in E. coli and purified on a nickel affinity column by standard biotechnology procedures.
  • the refolding was performed according to Raman S. et al., Nanomedicine (supra).
  • the refolded SAPN were analyzed for nanoparticle formation by dynamic light scattering (DLS) techniques and transmission electron microscopy (TEM).
  • the DLS analysis showed a nice size distribution with an average particle diameter of 46.96 nm and very low polydispersity index of 8.7%.
  • the TEM pictures ( FIG. 5 ) show nanoparticles of the size of about 30 nm.
  • This coiled-coil sequence contains two consecutive HTL epitopes with the sequences IRHENRMVL (SEQ ID NO:228) and YKIFKIEKG (residues 18-26 of SEQ ID NO:11) from the proteins M1 and neuraminidase of the influenza A virus, respectively.
  • IRHENRMVL SEQ ID NO:2278
  • YKIFKIEKG YKIFKIEKG
  • the first T-cell epitope IRHENRMVL (SEQ ID NO:228) is predicted to bind to many other HLA molecules as well such as B14, B1510, B2705, B2706, B3909, DP9, DR11, DR12, DR17, DR53, and DRB1, i.e. it is a predicted promiscuous epitope.
  • This trimeric coiled-coil was then included into the SAPN sequence SEQ ID NO:12 below, composed of a His-tag, the pentameric Trp-zipper coiled-coil, the trimeric coiled-coil as described above and the B-cell epitope from the CS-protein of Plasmodium berghei .
  • the peptide with this sequence was expressed in E. coli and purified on a nickel affinity column by standard biotechnology procedures.
  • the refolding was performed according to Raman S. et al., Nanomedicine (supra).
  • the refolded SAPN were analyzed for nanoparticle formation by dynamic light scattering (DLS) techniques and transmission electron microscopy (TEM).
  • the DLS analysis showed a somewhat broader size distribution with an average particle diameter of 57.70 nm and polydispersity index of 21.6%.
  • the TEM pictures ( FIG. 6 ) show nanoparticles of the size of about 30-50 nm.
  • cancer specific T-cell epitopes can be included into the SAPN forming peptide.
  • 19 different T-cell epitopes are engineered into the trimeric coiled-coil of the SAPN and the corresponding coiled-coil propensities are calculated for the first 10 of these sequences. It is nicely visible that the coiled-coil propensities for the T-cell epitopes are rather low, but the flanking sequences with very high coiled-coil propensities will compensate and induce coiled-coil formation throughout the whole sequence similar as shown for the examples 1 to 3 above.
  • This coiled-coil sequence contains the anti-parallel beta-turn peptide, which is the tip of the V3 loop of gp120 from HIV.
  • This peptide is a well-known B-cell epitope of HIV. It is inserted into the coiled-coil heptad repeat by two glycine residues at positions aa(b) and aa(c) of the coiled-coil.
  • flanking parts of the coiled-coil trimer are relatively short sequences but have strong coiled-coil forming propensity.
  • COILS coiled-coil prediction program
  • a peptide of the following sequence (SEQ ID NO:33) is recombinantly expressed in a standard E. coli expression system using a His-tag affinity purification scheme:
  • This sequence is related to the sequences from Examples 1 and 2 (SEQ ID NO:8 and SEQ ID NO:10), but without the C-terminal B-cell epitope.
  • the SAPN formed do not have a disulfide bridge between the two helices (underlined residues 55 and 64, replacement of the two cysteines by alanine as compared to the original design of Raman S. et al., Nanomedicine: Nanotechnology, Biology, and Medicine 2006; 2:95-102) but rather have the smaller amino acid alanine instead, allowing for smaller angles between the two helices, and hence more than 60 peptide chains are incorporated into the SAPN.
  • the molecular weight (MW) of the SAPN was assessed by analytical ultracentrifugation: The peptide was dissolved at 0.42 mg/ml, 0.34 mg/ml, and 0.21 mg/ml, in 150 mM NaCl, 20 mM Tris, pH 7.5. The measured MW corresponds to SAPN composed of about 330 monomers, i.e. a nanoparticle with more monomers than needed for a regular polyhedron with 60 asymmetric units (Table 23).
  • the two helices of the two oligomerization domains are not fixed by a disulfide bridge in their relative orientation to each other and the smaller amino acid alanine allows the two helices to get closer and hence the angle between them to be smaller.
  • the measured binding affinities of the peptides contained in the sequence LQSLTNLLSSNLSWLSLDVSAAF (residues 16-38 of SEQ ID NO:34) from the Hepatitis B Virus polymerase for the different MHCII molecules are as follows (binding affinities in nM in brackets): DRB1*0101 (2), DRB1*0301 (62), DRB1*0401 (10), DRB1*0405 (17), DRB1*0701 (173), DRB1*0802 (598), DRB1*0901 (791), DRB1*1101 (303), DRB1*1201 (397), DRB1*1302 (143), DRB1*1501 (21), DRB3*0101 (5), DRB4*0101 (7).
  • the sequence of the peptide is predicted to form a coiled-coil by the prediction program COILS.
  • the coiled-coil forming probability is more than 98% for all the residues in the sequence (Table 24), therefore the whole sequence is predicted to form a fully folded coiled-coil.
  • the following is an example of a trimeric coiled-coil design that includes a subsection of the HTL epitopes from SEQ ID NO:34 from (Mizukoshi E. et al., J Immunol 2004, 173:5863-5871) in combination with a sequence that is a subsection of the PADRE HTL epitope sequence.
  • binding affinities as predicted by the algorithm NetMHCII of the epitopes contained in the sequence ARFVAAWTLKVREVERELSWLSLDVSAAF (residues 17-45 of SEQ ID NO:35) for the different MHCII molecules as follows (binding affinities in nM in brackets): DRB1*0101 (23), DRB1*0401 (72), DRB1*0405 (37), DRB1*0701 (164), DRB1*0802 (462), DRB1*0901 (440), DRB1*1101 (271), DRB1*1302 (303), DRB1*1501 (16), DRB3*0101 (60), DRB4*0101 (51).
  • sequence pan3m contains in some part the same epitopes as the sequence pan3m (SEQ ID NO:34), which have in fact shown even stronger binding to the MHCII molecules than predicted by the NetMHCII program (Mizukoshi E. et al., J Immunol 2004, 173:5863-5871).
  • the sequence of the peptide is predicted to form a coiled-coil by the prediction program COILS.
  • the coiled-coil forming probability is more than 80% for all the residues in the sequence (Table 25) except for the last 8 residues, therefore the sequence is predicted to form a fully folded coiled-coil with the C-terminal end fraying a little bit apart, which will not interfere with SAPN formation as the majority of the sequence at N-terminal end form a coiled-coil.
  • the tetrameric coiled-coil sequence from tetrabrachion (Stetefeld J. et al., Nature Structural Biology 2000, 7(9):772-776) is characterized by an undecad coiled-coil repeat rather than a heptad repeat.
  • the following is a slightly modified sequence derived from this tetrameric coiled-coil.
  • This tetrameric coiled-coil is particularly well-suited as core coiled-coil of the SAPN as it contains a series of overlapping predicted HTL epitopes.
  • the sequences of the epitopes YRLTVIIDD (residues 2-10 of SEQ ID NO:36), LKNLITLRA (residues 15-23 of SEQ ID NO:36), LITLRADRL (residues 18-26 of SEQ ID NO:36), IINDNVSTLR (residues 29-38 of SEQ ID NO:36), INDNVSTLRA (residues 30-39 of SEQ ID NO:36), and VSTLRALLM (residues 34-42 of SEQ ID NO:36) are predicted by the algorithm NetMHCII to bind to the different MHC II molecules DRB1*0101, DRB1*0401, DRB1*0404, DRB1*0405, DRB1*0701, DRB1
  • the tetrameric coiled-coil is also well-suited to present tetrameric B-cell epitopes such as the M2e peptide from influenza, which has been done in the following SAPN design for a human vaccine with the sequence (SEQ ID NO:37):
  • the peptide contains starting from the N-terminus: the His-tag, the M2e B-cell epitope from a human-specific influenza strain, the tetrameric coiled-coil with the HTL epitopes, the linker, and the trimeric coiled-coil. With this sequence it was expressed in E. coli and purified on a nickel affinity column by standard biotechnology procedures. The refolding was performed according to Raman S. et al., Nanomedicine: Nanotechnology, Biology, and Medicine 2006; 2:95-102. The refolded SAPN were analyzed for nanoparticle formation by transmission electron microscopy (TEM). The TEM pictures ( FIG. 7A ) show nanoparticles of the same of about 30 nm.
  • chicken-specific M2e peptide from influenza can be displayed in its native oligomerization state and conformation as tetramer on the tetrameric coiled-coil of tetrabrachion, which has been done in the following SAPN design for an animal vaccine with the sequence (SEQ ID NO:38):
  • the peptide with this sequence (SEQ ID NO:38) was expressed in E. coli and purified on a nickel affinity column by standard biotechnology procedures. It contains starting from the N-terminus: the His-tag, the M2e B-cell epitope from a chicken-specific influenza strain, the tetrameric coiled-coil with the HTL epitopes, the linker, and the trimeric coiled-coil. The refolding was performed according to Raman S. et al., Nanomedicine: Nanotechnology, Biology, and Medicine 2006; 2:95-102 with the buffer of 20 mM Tris-HCl pH 7.5, 150 mM NaCl and 5% glycerol.
  • the refolded SAPN were analyzed for nanoparticle formation by dynamic light scattering (DLS) techniques and transmission electron microscopy (TEM).
  • the DLS analysis showed a size distribution with an average particle diameter of 45 nm and polydispersity index of 8.9%.
  • the TEM pictures ( FIG. 7B ) show nanoparticles of the same size of about 30 nm.
  • This sequence is predicted to form an ⁇ -helix (H) with high probability (mostly the highest score 9) according to the secondary structure prediction program PSIPRED (The PSIPRED Protein Structure Prediction Server). Since the core positions aa(a) and aa(d) of the heptad repeat of the coiled-coil are mostly tryptophan residues, this sequence is predicted to form a pentameric oiled coil (Liu J et al., Proc Natl Acad Sci USA 2004; 101(46):16156-61).
  • This pentameric coiled-coil sequence contains partly overlapping HTL epitopes with the sequences FVAAWTLKV (residues 3-11 of SEQ ID NO:39) WKIWKSLWK (residues 18-26 of SEQ ID NO:39) and KSLWKAWRL (residues 22-30 of SEQ ID NO:39) that are predicted by the algorithm NetMHCII to bind to the different MHC II molecules DRB1*0101, DRB1*0401, DRB1*0405, DRB1*0701, DRB1*0801, DRB1*0901, DRB1*1101, DRB1*1501, and DRB5*0101, respectively, with predicted binding affinities (nM) of 24, 73, 13, 120, 42, 596, 396, 6, and 13, respectively.
  • the sequence corresponds to the 17 C-terminal amino acids with two cysteines replaced by alanines, also know as CS.T3 peptide (SEQ ID NO:40) from the circum sporozoite protein CS.
  • the sequence of the peptide is predicted to form a coiled-coil by the prediction program COILS.
  • the coiled-coil forming probability is more than 99% for all the residues in the sequence (Table 26), except for the last two amino acids. Therefore the whole sequence is predicted to form a fully folded coiled-coil.
  • This coiled-coil (SEQ ID NO:40) has been used for the design of a SAPN with the following sequence (SEQ ID NO:41)
  • This sequence contains a his-tag, a pentameric coiled-coil tryptophan zipper, a linker, the trimeric coiled-coil SEQ ID NO:40, and a Plasmodium falciparum B-cell epitope, which is a tetra-repeat (NANP) of the repetitive sequence of the same circum sporozoite protein CS.
  • the peptide with this sequence was expressed in E. coli and purified on a nickel affinity column by standard biotechnology procedures.
  • the refolding was performed according to Raman S. et al., Nanomedicine (supra).
  • the refolded SAPN were analyzed for nanoparticle formation by dynamic light scattering (DLS) techniques and transmission electron microscopy (TEM).
  • the DLS analysis at pH 6.5 showed a size distribution with an particle diameter of 44.6 nm and a polydispersity index of 19.6%.
  • the TEM pictures ( FIG. 8 ) show nanoparticles of the size of about 30 nm.
  • HIV Vaccine HTL, CTL, B-Cell Co-Assembly
  • the first seven CTL peptide-strings of Table 10 are engineered at the C-terminal end of the six peptide chains with identical core and identical N-terminal B-cell epitope and hence are co-assembled into a single SAPN.
  • the core contains the same trimer as in Example 11 with a promiscuous P. falciparum HTL epitope, linked to a Trp-zipper pentameric coiled-coil which contains a PADRE HTL epitope.
  • the B-cell epitope is the membrane proximal region of GP41, which has neutralizing potential as evidenced by binding of the monoclonal neutralizing antibodies 2F5 and 4E10.
  • This epitope is ⁇ -helical in solution and is held in ⁇ -helical conformation by being partly engineered into the pentameric coiled-coil.
  • the surface accessible residues i.e. the residues that are not the coiled-coil core residues
  • the core is a combination of a trimeric coiled-coil that contains a CTL epitope MEKLKELEK (residues 75-83 of SEQ ID NO:53) and the modified B-cell epitope KLRNLEEELHSLRKNLNILNEELEELT (residues 84-110 of SEQ ID NO:53) (sequence 27 in Villard V. et al., PLoS ONE 2007, 2(7):e645), and the pentamer shown in Example 10 with excellent panDR binding properties.
  • B-cell epitope which is a tetra-repeat (NANP) of the repetitive sequence of the circum sporozoite protein CS from Plasmodium falciparum .
  • N-cell epitope a tetra-repeat
  • P. falciparum CTL epitopes U.S. Pat. Nos. 5,028,425, 5,972,351, 6,663,871
  • the CTL-epitopes are separated by optimized proteasomal cleavage sites (Hadeler K. P. et al., Math. Biosci. 2004, 188:63-79). Since the core of these six peptide chains is identical, co-assembly of these six peptide chains into one single SAPN allows the incorporation of about 18 different CTL epitopes into one single SAPN.
  • Influenza Vaccine HTL-Core, B-Cell Tetramer, CTL, Co-Assembly
  • the core (underlined) is a combination of the trimer in Example 7 and the tetramer in Example 9 with excellent panDR binding properties.
  • the tetrameric N-terminal B-cell epitope is the same as in Example 9.
  • the conserved CTL epitopes from Parida R. et al., Vaccine 2007, 25:7530-7539 (Table 15) are placed. Since the core of these six peptide chains is identical, co-assembly of these six peptide chains into one single SAPN allows the incorporation of about 20 different CTL epitopes into one single SAPN.
  • Influenza Vaccine HTL-Core, B-Cell Tetramer, Cleavage Peptide Co-Assembly
  • Influenza vaccine SAPN composed of three peptide chains with an identical core and identical N-terminal B-cell epitope M2e and nine B-cell epitopes at the C-terminus (three each per peptide chain) co-assembled into a single SAPN.
  • the core (underlined) is a combination of the trimer in Example 8 and the tetramer in Example 9 with excellent pan DR binding properties.
  • the tetrameric N-terminal B-cell epitope is the same as in Example 9.
  • the C-terminal B-cell epitopes are from Table 8 for H1, H2, H3, H5 consensus 1, H5 consensus 2, H7 consensus 1, H7 consensus 2, H7 consensus 3, H9 with negative charges between the epitopes to make the B-cell epitope string less positively charged.

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US11236134B2 (en) 2016-05-02 2022-02-01 The Scripps Research Institute Nucleic acids encoding HIV-1 GP140 immunogens comprising modified NHR1 regions that stabilize pre-fusion conformations
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JP2011511773A (ja) 2011-04-14
AU2009221419A1 (en) 2009-09-11
JP6088123B2 (ja) 2017-03-01
CA2713879A1 (fr) 2009-09-11
US20110020378A1 (en) 2011-01-27

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